Platform for the differentiation of cells

ABSTRACT

The present invention relates to an in vitro method for islet cell expansion, which comprises the steps of: a) preparing dedifferentiated cells derived from cells in or associated with post-natal islets of Langerhans; b) expanding the dedifferentiated cells; and c) inducing islet cell differentiation the expanded cells of step b) to become insulin-producing cells.

This application claims the benefit of provisional application No.60/118,790, filed Feb. 4, 1999.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to an in vitro method for islet cell expansion; anin vitro method for producing multi bipolar cells; an in vitro methodfor stem cell expansion; and a method for the treatment of diabetesmellitus in a patient.

(b) Description of Prior Art

Diabetes Mellitus

Diabetes mellitus has been classified as type I, or insulin-dependentdiabetes mellitus (IDDM) and type II, or non-insulin-dependent diabetesmellitus (NIDDM). NIDDM patients have been subdivided further into (a)nonobese (possibly IDDM in evolution), (b) obese, and (c) maturity onset(in young patients). Among the population with diabetes mellitus, about20% suffer from IDDM. Diabetes develops either when a diminished insulinoutput occurs or when a diminished sensitivity to insulin cannot becompensated for by an augmented capacity for insulin secretion. Inpatients with IDDM, a decrease in insulin secretion is the principalfactor in the pathogenesis, whereas in patients with NIDDM, a decreasein insulin sensitivity is the primary factor. The mainstay of diabetestreatment, especially for type I disease, has been the administration ofexogenous insulin.

Rationale for More Physiologic Therapies

Tight glucose control appears to be the key to the prevention of thesecondary complications of diabetes. The results of the DiabetesComplications and Control Trial (DCCT), a multicenter randomized trialof 1441 patients with insulin dependent diabetes, indicated that theonset and progression of diabetic retinopathy, nephropathy, andneuropathy could be slowed by intensive insulin therapy (The DiabetesControl and Complication Trial Research Group, N. Engl. J. Med., 1993;29:977-986). Strict glucose control, however, was associated with athree-fold increase in incidence of severe hypoglycemia, includingepisodes of seizure and coma. As well, although glycosylated hemoglobinlevels decreased in the treatment group, only 5% maintained an averagelevel below 6.05% despite the enormous amount of effort and resourcesallocated to the support of patients on the intensive regime (TheDiabetes Control and Complication Trial Research Group, N. Engl. J.Med., 1993; 29:977-986). The results of the DCCT clearly indicated thatintensive control of glucose can significantly reduce (but notcompletely protect against) the long-term microvascular complications ofdiabetes mellitus.

Other Therapeutic Options

The delivery of insulin in a physiologic manner has been an elusive goalsince insulin was first purified by Banting, Best, McLeod and Collip.Even in a patient with tight glucose control, however, exogenous insulinhas not been able to achieve the glucose metabolism of an endogenousinsulin source that responds to moment-to-moment changes in glucoseconcentration and therefore protects against the development ofmicrovascular complications over the long term.

A major goal of diabetes research, therefore, has been the developmentof new forms of treatment that endeavor to reproduce more closely thenormal physiologic state. One such approach, a closed-loop insulin pumpcoupled to a glucose sensor, mimicking β-cell function in which thesecretion of insulin is closely regulated, has not yet been successful.Only total endocrine replacement therapy in the form of a transplant hasproven effective in the treatment of diabetes mellitus. Althoughtransplants of insulin-producing tissue are a logical advance oversubcutaneous insulin injections, it is still far from clear whether therisks of the intervention and of the associated long-termimmunosuppressive treatment are lower those in diabetic patients underconventional treatment.

Despite the early evidence of the potential benefits of vascularizedpancreas transplantation, it remains a complex surgical intervention,requiring the long-term administration of chronic immunosuppression withits attendant side effects. Moreover, almost 50% of successfullytransplanted patients exhibit impaired tolerance curves (Wright F H etal., Arch. Surg., 1989;124:796-799; Landgraft R et al., Diabetologia1991; 34 (suppl 1):S61; Morel P et al., Transplantation 1991;51:990-1000), raising questions about their protection against thelong-term complications of chronic hyperglycemia.

The major complications of whole pancreas transplantation, as well asthe requirement for long term immunosuppression, has limited its widerapplication and provided impetus for the development of islettransplantation. Theoretically, the transplantation of islets alone,while enabling tight glycemic control, has several potential advantagesover whole pancreas transplantation. These include the following: (i)minimal surgical morbidity, with the infusion of islets directly intothe liver via the portal vein; (ii) the possibility of simplere-transplantation for graft failures; (iii) the exclusion ofcomplications associated with the exocrine pancreas; (iv) thepossibility that islets are less immunogenic, eliminating the need forimmunosuppression and enabling early transplantation into non-uremicdiabetics; (v) the possibility of modifying islets in vitro prior totransplantation to reduce their immunogenicity; (vi) the ability toencapsulate islets in artificial membranes to isolate them from the hostimmune system; and (vii) the related possibility of usingxenotransplantation of islets immunoisolated as part of a biohybridsystem. Moreover, they permit the banking of the endocrine cryopreservedtissue and a careful and standardized quality control program before theimplantation.

The Problem of Islet Transplantation

Adequate numbers of isogenetic islets transplanted into a reliableimplantation site can only reverse the metabolic abnormalities indiabetic recipients in the short term. In those that were normoglycemicpost-transplant, hyperglycemia recurred within 3-12 mo. (Orloff M, et.al., Transplantation 1988; 45:307). The return of the diabetic statethat occurs with time has been attributed either to the ectopic locationof the islets, to a disruption of the enteroinsular axis, or to thetransplantation of an inadequate islet cell mass (Bretzel R G, et al.In: Bretzel R G, (ed) Diabetes mellitus. (Berlin: Springer, 1990)p.229).

Studies of the long term natural history of the islet transplant, thatexamine parameters other than graft function, are few in number. Onlyone report was found in which an attempt was specifically made to studygraft morphology (Alejandro R, et. al., J Clin Invest 1986; 78: 1339).In that study, purified islets were transplanted into the canine livervia the portal vein. During prolonged follow-up, delayed failures ofgraft function occurred. Unfortunately, the graft was only examined atthe end of the study, and not over time as function declined. Delayedgraft failures have also been confirmed by other investigators for dogs(Warnock G L et. al., Can. J. Surg., 1988; 31: 421 and primates (SuttonR, et. al., Transplant Proc., 1987; 19: 3525). Most failures arepresumed to be the result of rejection despite appropriateimmunosuppression.

Because of these failures, there is currently much enthusiasm for theimmunoisolation of islets, which could eliminate the need forimmunosuppression. The reasons are compelling. Immunosuppression isharmful to the recipient, and may impair islet function and possiblycell survival (Metrakos P, et al., J. Surg. Res., 1993; 54: 375).Unfortunately, micro-encapsulated islets injected into the peritonealcavity of the dog fail within 6 months (Soon-Shiong P, et. al.,Transplantation 1992; 54: 769), and islets placed into a vascularizedbiohybrid pancreas also fail, but at about one year. In each instance,however, histological evaluation of the graft has indicated asubstantial loss of islet mass in these devices (Lanza R P, et. al.,Diabetes 1992; 41: 1503). No reasons have been advanced for thesechanges. Therefore maintenance of an effective islet cell masspost-transplantation remains a significant problem.

In addition to this unresolved issue, is the ongoing problem of the lackof source tissue for transplantation. The number of human donors isinsufficient to keep up with the potential number of recipients.Moreover, given the current state of the art of islet isolation, thenumber of islets that can be isolated from one pancreas is far from thenumber required to effectively reverse hyperglycemia in a humanrecipient.

In response, three competing technologies have been proposed and areunder development. First, islet cryopreservation and islet banking. Thetechniques involved, though, are expensive and cumbersome, and do noteasily lend themselves to widespread adoption. In addition, islet cellmass is also lost during the freeze-thaw cycle. Therefore this is a poorlong-term solution to the problem of insufficient islet cell mass.Second, is the development of islet xenotransplantation. This idea hasbeen coupled to islet encapsulation technology to produce a biohybridimplant that does not, at least in theory, require immunosuppression.There remain many problems to solve with this approach, not least ofwhich, is that the problem of the maintenance of islet cell mass in thepost-transplant still remains. Third, is the resort to human fetaltissue, which should have a great capacity to be expanded ex vivo andthen transplanted. However, in addition to the problems of limitedtissue availability, immunogenicity, there are complex ethical issuessurrounding the use of such a tissue source that will not soon beresolved. However, there is an alternative that offers similarpossibilities for near unlimited cell mass expansion.

An entirely novel approach, proposed by Rosenberg in 1995 (Rosenberg Let al., Cell Transplantation, 1995;4:371-384), was the development oftechnology to control and modulate islet cell neogenesis and new isletformation, both in vitro and in vivo. The concept assumed that (a) theinduction of islet cell differentiation was in fact controllable; (b)implied the persistence of a stem cell-like cell in the adult pancreas;and (c) that the signal(s) that would drive the whole process could beidentified and manipulated.

In a series of in vivo studies, Rosenberg and co-workers establishedthat these concepts were valid in principle, in the in vivo setting(Rosenberg L et al., Diabetes, 1988;37:334-341; Rosenberg L et al.,Diabetologia, 1996;39:256-262), and that diabetes could be reversed.

The well known teachings of in vitro islet cell expansion from anon-fetal tissue source comes from Peck and co-workers (Corneliu J G etal., Horm. Metab. Res., 1997;29:271-277), who describe isolation of apluripotent stem cell from the adult mouse pancreas that can be directedtoward an insulin producing cell. These findings have not been widelyaccepted. First, the result has not proven to be reproducible. Second,the so-called pluripotential cells have never been adequatelycharacterized with respect to phenotype. And third, the cells havecertainly not been shown to be pluripotent.

More recently two other competing technologies have been proposed theuse of engineered pancreatic β-cell lines (Efrat S, Advanced DrugDelivery Reviews, 1998;33:45-52), and the use of pluripotent embryonalstem cells (Shamblott M J et al., Proc. Natl. Acad. Sci. USA,1998;95:13726-13731). The former option, while attractive, is associatedwith significant problems. Not only must the engineered cell be able toproduce insulin, but it must respond in a physiologic manner to theprevailing level of glucose—and the glucose sensing mechanism is farfrom being understood well enough to engineer it into a cell. Manyproposed cell lines are also transformed lines, and therefore have aneoplastic potential. With respect to the latter option, having anembryonal stem cell in hand is appealing because of the theoreticalpossibility of being able to induce differentiation in any direction,including toward the pancreatic β-cell. However, the signals necessaryto achieve this milestone remain unknown.

It would be highly desirable to be provided with a platform for thepreparation of dedifferentiated cells derived from post-natal islets ofLangerhans, their expansion and the guided induction of islet celldifferentiation, leading to insulin-producing cells that can be used forthe treatment of diabetes mellitus.

SUMMARY OF THE INVENTION

One aim of the invention is to provide a platform for the preparation ofdedifferentiated cells derived from cells in or associated withpost-natal islets of Langerhans, their expansion and the guidedinduction of islet cell differentiation, leading to insulin-producingcells that can be used for the treatment of diabetes mellitus.

In accordance with one embodiment of the present invention there isprovided an in vitro method for islet cell expansion, which comprisesthe steps of:

a) preparing dedifferentiated cells derived from cells in or associatedwith post-natal islets of Langerhans;

b) expanding the dedifferentiated cells; and

c) inducing islet cell differentiation of the expanded cells of step b)to become insulin-producing cells.

Preferably, step a) and step b) are concurrently effected using a solidmatrix, basal feeding medium and appropriate growth factors to permitthe development, maintenance and expansion of a dedifferentiated cellpopulation with at least bipotentiality or being multipotent.

Preferably, step c) is effected by removing cells from the matrix andresuspended in a basal liquid medium containing soluble matrix proteinsand growth factors.

Preferably, the basal liquid medium is CMRL 1066 supplemented with 10%fetal calf serum, wherein the soluble matrix proteins and growth factorsare selected from the group consisting of fibronectin, IGF -1, IGF-2,insulin, and NGF. The basal liquid medium may further comprise glucoseconcentration of at least 11 mM. The basal liquid medium may furthercomprise inhibitors of known intracellular signaling pathways ofapoptosis and/or specific inhibitor of p38.

In accordance with another embodiment of the present invention there isprovided an in vitro method for producing cells with at leastbipotentiality, which comprises the steps of:

a) preparing dedifferentiated cells derived from cells in or associatedwith post-natal islets of Langerhans from a patient; whereby when thededifferentiated cells are introduced in situ in the patient, the cellsare expanded and undergo islet cell differentiation to become in situinsulin-producing cells.

In accordance with another embodiment of the present invention there isprovided a method for the treatment of diabetes mellitus in a patient,which comprises the steps of

a) preparing dedifferentiated cells derived from cells in or associatedwith post-natal islets of Langerhans of the patient; and

b) introducing the dedifferentiated cells in situ in the patient,wherein the cells expand in situ and undergo islet cell differentiationin situ to become insulin-producing cells.

In accordance with another embodiment of the present invention there isprovided a method for the treatment of diabetes mellitus in a patient,which comprises the steps of

a) preparing dedifferentiated cells derived from cells in or associatedwith post-natal islets of Langerhans of the patient;

b) expanding in vitro the dedifferentiated cells;

c) inducing in vitro islet cell differentiation of the expanded cells ofstep b) to become insulin-producing cells; and

d) introducing the cells of step c) in situ in the patient, wherein thecells produce insulin in situ.

For the purpose of the present invention the following terms are definedbelow.

The expression “post-natal islets of Langerhans” is intended to meanislet cells and associated cells, such as duct cells, of any origin,such as human, porcine and canine, among others.

The expression “dedifferentiated cells” is intended to mean cells of anyorigin which are stem-like cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 illustrates cell-type conversion from Islet to duct-likestructure (human tissues), (a) let in the pancreas, (b) Islet followingisolation and purification, (c) islet in solid matrix beginning toundergo cystic change, (d-f) progressive formation of cystic structurewith complete loss of islet morphology.

FIG. 2 illustrates same progression of changes as in FIG. 1. Cells arestained by immunocytochemistry for insulin. (a) Islet in pancreas. (b)Islet after isolation and purification. (c) Islet in solid matrixbeginning to undergo cystic change. (d-e) Progressive loss of isletphenotype. (f) High power view of cyst wall composed duct-likeepithelial cells. One cell still contains insulin (arrow).

FIG. 3 illustrates same progression of changes as in FIG. 1. Cellsstained by immunocytochemistry for glucagon. (a) Islet in pancreas. (b)Islet after isolation and purification. (c) Islet in solid matrixbeginning to undergo cystic change. (d-e) Progressive loss of isletphenotype. (f) High power view of cyst wall composed duct-likeepithelial cells. One cell still contains glucagon (arrow).

FIG. 4 illustrates demonstration of cell phenotype by CK-19immunocytochemistry. Upper left panel—cystic structure in solid matrix.All cells stain for CK-19, a marker expressed in ductal epithelial cellsin the pancreas. Lower panel—following removal from the solid matrix,and return to suspension culture. A structure exhibiting bothepithelial-like and solid components. Upper right panel—only theepithelial-like component retains CK-10 immunoreactivity. The solidcomponent has lost its CK -19 expression, and appears islet-like.

FIG. 5 illustrates upper panel—Ultrastructural appearance of cellscomposing the cystic structures in solid matrix. Note the microvilli andloss of endosecretory granules. The cells have the appearance ofprimitive duct-like cells. Lower panel—ultrastructural appearance ofcystic structures removed from the solid matrix and placed in suspensionculture. Note the decrease in microvilli and the reappearance ofendosecretory granules.

FIG. 6 illustrates in situ hybridization for pro-insulin mRNA. Upperpanel—cystic structures with virtually no cells containing the message.Lower panel—cystic structures have been removed from the matrix andplaced in suspension culture. Note the appearance now, of both solid andcystic structures. The solid structures have an abundant expression ofpro-insulin mRNA.

FIG. 7 illustrates insulin release into the culture medium by thestructures seen in the lower panel of FIG. 6. Note that there is nodemonstrable insulin secreted from the tissue when in the cystic state(far left column). FN-fibronectin; IGF-1-insulin-like growth factor-1;Gluc-glucose.

DETAILED DESCRIPTION OF THE INVENTION

In vivo cell transformation leading to β-cell neogenesis and new isletformation can be understood in the context of established concepts ofdevelopmental biology.

Transdifferentiation is a change from one differentiated phenotype toanother, involving morphological and functional phenotypic markers(Okada T S., Develop. Growth and Differ. 1986;28:213-321). Thebest-studied example of this process is the change of amphibian iridialpigment cells to lens fibers, which proceeds through a sequence ofcellular dedifferentiation, proliferation and finally redifferentiation(Okada T S, Cell Diff. 1983;13:177-183; Okada T S, Kondoh H, Curr. TopDev. Biol., 1986;20:1-433; Yamada T, Monogr. Dev. Biol., 1977;13:1-124).Direct transdifferentiation without cell division has also beenreported, although it is much less common (Beresford W A, Cell Differ.Dev., 1990;29:81-93). While transdifferentiation has been thought to beessentially irreversible, i.e. the transdifferentiated cell does notrevert back into the cell type from which it arose, this has recentlybeen reported not to be the case (Danto S I et al., Am. J. Respir. CellMol. Biol., 1995;12:497-502). Nonetheless, demonstration oftransdifferentiation depends on defining in detail the phenotype of theoriginal cells, and on proving that the new cell type is in factdescended from cells that were defined (Okada T S, Develop. Growth andDiffer. 1986;28:213-321).

In many instances, transdifferentiation involves a sequence of steps.Early in the process, intermediate cells appear that express neither thephenotype of the original nor the subsequent differentiated cell types,and therefore they have been termed dedifferentiated. The whole processis accompanied by DNA replication and cell proliferation.Dedifferentiated cells are assumed a priori to be capable of formingeither the original or a new cell type, and thus are multipotential(Itoh Y, Eguchi G, Cell Differ., 1986;18:173-182; Itoh Y, Eguchi G,Develop. Biology, 1986;115:353-362; Okada T S, Develop. Growth andDiffer, 1986;28:213-321).

Stability of the cellular phenotype in adult organisms is probablyrelated to the extracellular milieu, as well as cytoplasmic and nuclearcomponents that interact to control gene expression. The conversion ofcell phenotype is likely to be accomplished by selective enhancement ofgene expression, which controls the terminal developmental commitment ofcells.

The pancreas is composed of several types of endocrine and exocrinecells, each responding to a variety of trophic influences. The abilityof these cells to undergo a change in phenotype has been extensivelyinvestigated because of the implications for the understanding ofpancreatic diseases such as cancer and diabetes mellitus.Transdifferentiation of pancreatic cells was first noted nearly a decadeago. Hepatocyte-like cells, which are normally not present in thepancreas, were observed following the administration of carcinogen (RaoM S et al., Am. J. Pathol., 1983;110:89-94; Scarpelli D G, Rao M S,Proc. Nat. Acad. Sci. USA 1981;78:2577-2581) to hamsters and the feedingof copper-depleted diets to rats (Rao M S, et al., Cell Differ.,1986;18:109-117). Recently, transdifferentiation of isolated acinarcells into duct-like cells has been observed by several groups (Arias AE, Bendayan M, Lab Invest., 1993;69:518-530; Hall P A, Lemoine N R, J.Pathol., 1992;166:97-103; Tsao M S, Duguid W P, Exp. Cell Res.,1987;168:365-375). In view of these observations it is probably germanethat during embryonic development, the hepatic and pancreatic anlagenare derived from a common endodermal.

An alternative to transdifferentiation, is the possibility that newislet cells arise from stem cells that persist post-natally in adulttissue.

There are two general categories of stem cells (Young H E et al. PSEBM1999; 221:63-71; Young H E et al. Wound Rep Regen 1998; 6:65-75).Progenitors are (a) lineage committed (i.e. they will form only tissueswithin their respective committed lineages(s)); (b) they prefer toremain quiescent and therefore need to be actively stimulated orchallenged to do anything; (c) their life-span is approx. 50-70 celldivisions before programmed cell death intervenes; (d) they areunresponsive to inductive agents outside their lineage; and (e) they areresponsive to progression agents (e.g. insulin, IGF-1 or IGF-2) whichare needed to promote phenotypic expression into lineage restrictedphenotypes only. Pluripotents, on the other hand, are lineageuncommitted, derived from the inner cell mass of the blastocyst.

Within these two broad categories, there are four types of cells—(1) thetotipotent stem cell; (2) the pluripotent stem cell; (3) the multipotentstem cell, and (4) the unipotent stem cell. Multipotent cells (committedto two or more cell lineage, e.g. chondro-osteogenic, adipo-fibrogenic)and unipotent cells (committed to a single tissue lineage, e.g.myogenic, adipogenic, osteogenic), are considered to be progenitorcells. To date, progenitor cells have been identified from six speciesthus far, and also from fetal to geriatric aged individuals. It is quitepossible, therefore, that islet cell differentiation post-natally mayoccur as a result of the stimulation of a unipotent or multipotentprogenitor cell as opposed to transdifferentiation.

One example of such a mechanism can be observed in the liver. Hepaticoval cells are a small sub-population of cells found in the liver whenhepatocyte proliferation is inhibited and followed by some type ofhepatic injury. They are believed to be bipotential, able todifferentiate into into hepatocytes or bile duct epithelium. Theyexpress the same markers as hematopoietic stem cells (HSC), and evidencehas been obtained that these cells can be derived from a bone marrowsource (Petersen B E, et al. Science 1999;284:1168-1170). In thiscontext, it is quite possible that the hepatocyte-like cells identifiedin the pancreas, to which we referred above (Rao M S et al., Am. J.Pathol., 1983;110:89-94; Scarpelli D G, Rao M S, Proc. Nat. Acad. Sci.USA 1981;78:2577-2581; Rao M S, et al., Cell Differ., 1986;18:109-117),may have in fact been derived from the equivalent of oval cells in thepancreas.

Factors which control the growth and functional maturation of the humanendocrine pancreas during the fetal and post-natal periods are stillpoorly understood, although the presence of specific factors in thepancreas has been hypothesized (Pictet R L et al. In: ExtracellularMatrix Influences on Gene Expression. Slavkin H C, Greulich R C (eds).Academic Press, New York, 1975, pp.10).

Some information is available on exocrine growth factors. MesenchymalFactor (MF), has been extracted from particulate fractions ofhomogenates of midgestational rat or chick embryos. MF affects celldevelopment by interacting at the cell surface of precursor cells(Rutter W J. The development of the endocrine and exocrine pancreas. In:The Pancreas. Fitzgerald P J, Morson A B (eds). Williams and Wilkins,London, 1980, pp.30) and thereby influences the kind of cells thatappear during pancreatic development (Githens S. Differentiation anddevelopment of the exocrine pancreas in animals. In: Go V L W, et al.(eds) The Exocrine Pancreas: Biology, Pathobiology and Diseases. RavenPress, New York, 1986, pp.21). MF is comprised of at least 2 fundamentalcomponents, a heat stable component whose action can be duplicated bycyclic AMP analogs, and another high molecular weight protein component(Rutter W J, In: The Pancreas. Fitzgerald P J, Morson A B (eds).Williams and Wilkins, London, 1980, pp.30). In the presence of MF, cellsdivide actively and differentiate largely into non-endocrine cells.

Other factors have also been implicated in endocrine maturation. Solublepeptide growth factors (GF) are one group of trophic substances thatregulate both cell proliferation and differentiation. These growthfactors are multi-functional and may trigger a broad range of cellularresponses (Sporn & Roberts, Nature, 332:217-19, 1987). Their actions canbe divided into 2 general categories—effects on cell proliferation,which comprises initiation of cell growth, cell division and celldifferentiation; and effects on cell function. They differ from thepolypeptide hormones in that they act in an autocrine and/or paracrinemanner (Goustin A S, Leof E B, et al. Cancer Res., 46:1015-1029, 1986;Underwood L E, et al., Clinics in Endocrinol. & Metabol.,15:59-77,1986). Specifics of their role in the individual processes thatcomprise growth need to be resolved.

One family of growth factors are the somatomedins. Insulin-like growthfactor-I (IGF-I), is synthesized and released in tissue culture by theβ-cells of fetal and neonatal rat islets (Hill D J, et al., Diabetes,36:465-471, 1987; Rabinovitch A, et al., Diabetes, 31:160-164,1982;Romanus J A et al., Diabetes 34:696-792, 1985). IGF-II has beenidentified in human fetal pancreas (Bryson J M et al., J. Endocrinol.,121:367-373,1989). Both these factors enhance neonatal β-cellreplication in vitro when added to the culture medium (Hill D J, et al.,Diabetes, 36:465-471, 1987; Rabinovitch A, et al., Diabetes, 31:160-164,1982). Therefore the IGF's may be important mediators of cellreplication in fetal and neonatal rat islets but may not do so inpost-natal development (Billestrup N, Martin J M, Endocrinol.,116:1175-81,1985; Rabinovitch A, et al., Diabetes, 32:307-12, 1983;Swenne I, Hill D J, Diabetologia 32:191-197, 1989; Swenne I,Endocrinology, 122:214-218, 1988; Whittaker P G, et al, Diabetologia,18:323-328, 1980). Furthermore, Platelet-derived growth factor (PDGF)also stimulates fetal islet cell replication and its effect does notrequire increased production of IGF-I (Swenne I, Endocrinology,122:214-218, 1988). Moreover, the effect of growth hormone on thereplication of rat fetal β-cells appears to be largely independent ofIGF -I (Romanus J A et al., Diabetes 34:696-792, 1985; Swenne I, Hill DJ, Diabetologia 32:191-197, 1989). In the adult pancreas, IGF-I mRNA islocalized to the D-cell. But IGF-I is also found on cell membranes of β-and A-cells, and in scattered duct cells, but not in acinar or vascularendothelial cells (Hansson H-A et al., Acta Physiol. Scand. 132:569-576,1988; Hansson H-A et al., Cell Tissue Res., 255:467-474, 1989). This isin contradistinction to one report (Smith F et al, Diabetes, 39 (suppl1):66A, 1990), wherein IGF-I expression was identified in ductular andvascular endothelial cells, and appeared in regenerating endocrine cellsafter partial pancreatectomy. It has not been shown that IGF's willstimulate growth of adult duct cells or islets. Nor do the IGF'sstimulate growth of the exocrine pancreas (Mossner J et al., Gut28:51-55, 1987). It is apparent therefore, that the role of IGF-I,especially in the adult pancreas, is far from certain.

Fibroblast growth factor (FGF) has been found to initiatetransdifferentiation of the retinal pigment epithelium to neural retinaltissues in chick embryo in vivo and in vitro (Hyuga M et al., Int. J.Dev. Biol. 1993;37:319-326; Park C M et al., Dev. Biol.1991;148:322-333; Pittack C et al., Development 1991;113:577-588).Transforming growth factor-beta (TGF-β) has been demonstrated to inducetransdifferentiation of mouse mammary epithelial cells to fibroblastcells [20]. Similarly, epithelial growth factor (EGF) and cholera toxinwere used to enhance duct epithelial cyst formation from amongpancreatic acinar cell fragments (Yuan S et al., In vitro Cell Dev.Biol., 1995;31:77-80).

The search for the factors mediating cell differentiation and survivalmust include both the cell and its microenvironment (Bissell M J et al.,J. Theor. Biol., 1982; 99:31), as a cell's behavior is controlled byother cells as well as by the extracellular matrix (ECM) (Stoker A W etal. Curr. Opin. Cell. Biol., 1990;2:864). ECM is a dynamic complex ofmolecules serving as a scaffold for parenchymal and nonparenchymalcells. Its importance in pancreatic development is highlighted by therole of fetal mesenchyme in epithelial cell cytodifferentiation(Bencosme S A, Am. J. Pathol. 1955; 31: 1149; Gepts W, de Mey J.Diabetes 1978; 27(suppl. 1): 251; Gepts W, Lacompte P M. Am. J. Med.,1981; 70: 105; Gepts W. Diabetes 1965; 14: 619; Githens S. In: Go V L W,et al. (eds) The Exocrine Pancreas: Biology, Pathobiology and Disease.(New York: Raven Press, 1986) p. 21). ECM is found in twoforms—interstitial matrix and basement membrane (BM). BM is amacromolecular complex of different glycoproteins, collagens, andproteoglycans. In the pancreas, the BM contains laminin, fibronectin,collagen types IV and V, as well as heparan sulphate proteoglycans(Ingber D. In: Go V L W, et al (eds) The Pancreas: Biology, Pathobiologyand Disease (New York: Raven Press, 1993) p. 369). The specific role ofthese molecules in the pancreas has yet to be determined.

ECM has profound effects on differentiation. Mature epithelia thatnormally never express mesenchymal genes, can be induced to do so bysuspension in collagen gels in vitro (Hay E D. Curr. Opin. in Cell.Biol. 1993; 5:1029). For example, mammary epithelial cells flatten andlose their differentiated phenotype when attached to plastic dishes oradherent collagen gels (Emerman J T, Pitelka D R. In vitro 1977;13:316). The same cells round, polarize, secrete milk proteins, andaccumulate a continuous BM when the gel is allowed to contract (EmermanJ T, Pitelka D R. In vitro, 1977; 13:316). Thus different degrees ofretention or re-formation of BM are crucial for cell survival and themaintenance of the normal epithelial phenotype (Hay E D. Curr. Opin. inCell. Biol. 1993; 5:1029).

During times of tissue proliferation, and in the presence of theappropriate growth factors, cells are transiently released fromECM-determined survival constraints. It is now becoming clear that thereare two components of the cellular response to ECM interactions—onephysical, involving shape changes and cytoskeletal organization; theother biochemical, involving integrin clustering and increased proteintyrosine phosphorylation (Ingber D E. Proc. Natl. Acad. Sci. USA,1990;87:3579; Roskelley C D et al., Proc. Natl. Acad. Sci. USA,1994;91:12378)

In addition to its known regulatory role in cellular growth anddifferentiation, ECM has more recently been recognized as a regulator ofcell survival (Bates R C, Lincz L F, Burns G F, Cancer and MetastasisRev., 1995;14:191). Disruption of the cell-matrix relationship leads toapoptosis (Frisch S M, Francis H. J. Cell. Biol., 1994;124:619; SchwartzS M, Bennett M R, Am. J. Path., 1995;147:229), a morphological series ofevents (Kerr J F K et al., Br. J. Cancer, 1972;26:239), indicating aprocess of active cellular self destruction.

In accordance with one embodiment of the present invention, the platformtechnology is based on a combination of the foregoing observations,incorporating the following components that are necessary and sufficientfor the preparation of dedifferentiated intermediate cells from adultpancreatic islets of Langerhans:

1. a solid matrix permitting “three dimensional” culture;

2. the presence of matrix proteins including but not limited to collagentype I and laminin; and

3. the growth factor EGF and promoters of cAMP, including but notlimited to cholera toxin and forskolin.

The preferred feeding medium is DMEM/F12 with 10% fetal calf serum. Inaddition, the starting tissue must be freshly isolated and culturedwithout absolute purification.

The use of a matrix protein-containing solid gel is an important part ofthe culture system, because extracellular matrix may promote the processof transdifferentiation. This point is highlighted by isolatedpancreatic acinar cells, which transdifferentiate to duct-likestructures when entrapped in Matrigel basement membrane (Arias A E,Bendayan M, Lab Invest., 1993;69:518-530), or by retinal pigmentedepithelial cells, which transdifferentiate into neurons when plated onlaminin-containing substrates (Reh T A et al., Nature 1987;330:68-71).Most recently, Gittes et al. demonstrated, using 11-day embryonic mousepancreas, that the default path for growth of embryonic pancreaticepithelium is to form islets (Gittes G K et al., Development 1996;122:439-447). In the presence of basement membrane constituents,however, the pancreatic anlage epithelium appears to programmed to formducts. This finding again emphasizes the interrelationship between ductsand islets and highlights the important role of the extracellularmatrix.

This completes stage 1 (the production of dedifferentiated intermediatecells) of the process. During the initial 96 h of culture, isletsundergo a cystic transformation associated with (Arias A E, Bendayan M,Lab. Invest., 1993;69:518-530) a progressive loss of insulin geneexpression, (2) a loss of immunoreactivity for insulin protein, and (3)the appearance of CKA 19, a marker for ductal cells. Aftertransformation is complete, the cells have the ultrastructuralappearance of primitive duct-like cells. Cyst enlargement after theinitial 96 h is associated, at least in part, with a tremendous increasein cell replication. These findings are consistent with thetransdifferentiation of an islet cell to a ductal cell (Yuan et al.,Differentiation, 1996; 61:67-75).

Stage 2—the generation of functioning β-cells, requires a completechange of the culture conditions. The cells are moved from the digestedmatrix and resuspended in a basal liquid medium such as CMRL 1066supplemented with 10% fetal calf serum, with the addition of solublematrix proteins and growth factors that include, but are not limited to,fibronectin (10-20 ng/ml), IGF-1 (100 ng/ml), IGF-2 (100 ng), insulin(10-100 μg/ml), NGF (10-100 ng/ml). In addition, the glucoseconcentration must be increased to above 11 mM. Additional cultureadditives may include specific inhibitors of known intracellularsignaling pathways of apoptosis, including, but not limited to aspecific inhibitor of p38.

Evidence for the return to an islet cell phenotype includes: (1) there-appearance of solid spherical structures; (2) loss of CK-19expression; (3) the demonstration of endosecretory granules on electronmicroscopy; (4) the re-appearance of pro-insulin mRNA on in situhybridization; (5) the return of a basal release of insulin into theculture medium.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. An in vitro method for islet cell expansion,which comprises the steps of: a) inducing cystic formation in cells inor associated with post-natal islets of Langerhans to obtain a duct-likestructure; b) expanding cells of said duct-like structure; and c)inducing islet cell differentiation of said expanded cells of saidduct-like structure to become insulin-producing cells; wherein step a)and step b) are concurrently effected using a solid matrix, basementmembrane constituents, basal feeding medium and appropriate growthfactors to permit the development, maintenance and expansion of aduct-like structure cell population with at least bipotentiality;wherein step c) is effected by removing cells from said matrix andresuspended in a basal liquid medium containing soluble matrix proteinsand growth factors selected from the group consisting of fibronectin,IGF-1, IGF-2, insulin, and NGF.
 2. The method of claim 1, wherein saidbasal liquid medium is CMRL 1066 supplemented with 10% fetal calf serum.3. The method of claim 1, wherein said basal liquid medium furthercomprises glucose concentration of at least 11 mM.
 4. The method ofclaim 3, wherein said basal liquid medium further comprises inhibitorsof known intracellular signaling pathways of apoptosis and/or specificinhibitor of p38.